Geomicrobiology Journal

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X-Ray Diffraction of Iron Containing Samples: The

Importance of a Suitable Configuration

Yvonne M. Mos, Arnold C. Vermeulen, Cees J. N. Buisman & Jan Weijma

To cite this article: Yvonne M. Mos, Arnold C. Vermeulen, Cees J. N. Buisman & Jan Weijma
(2018) X-Ray Diffraction of Iron Containing Samples: The Importance of a Suitable Configuration,
Geomicrobiology Journal, 35:6, 511-517, DOI: 10.1080/01490451.2017.1401183

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GEOMICROBIOLOGY JOURNAL, 2018
VOL. 35, NO. 6, 511–517
https://doi.org/10.1080/01490451.2017.1401183

X-Ray Diffraction of Iron Containing Samples: The Importance of a Suitable

Configuration
Yvonne M. Mosa, Arnold C. Vermeulenb, Cees J. N. Buismana, and Jan Weijmaa
a
Sub-Department of Environmental Technology, Wageningen University, Bornse Weilanden 9, Wageningen, The Netherlands;
b
PANalytical, Lelyweg 1, Almelo, The Netherlands

ABSTRACT ARTICLE HISTORY

In X-ray diffraction, a good combination of configuration and sample is essential. Copper radiation for iron Received 14 June 2016
containing materials leads to a high background. Although this has been recognized, many researchers Accepted 1 November 2017
still use this combination. To clearly show the unsuitability of copper radiation for iron oxides, magnetite, KEYWORDS
goethite, maghemite, and hematite were analysed in different configurations using copper or cobalt Fluorescence; iron; radiation
radiation. Results show effects of fluorescence repressing measures and different radiation sources. type; X-ray diffraction
Copper radiation diffractograms make phase identification contestable. Studies using copper radiation for
iron oxides must therefore be carefully evaluated. Cobalt radiation yielded high quality diffractograms,
making phase identification unambiguous.

Introduction
The iron redox cycle is important in the field of geomicrobiology.
Ferric and ferrous iron are abundant in nature and can be reduced
or oxidized, depending on the redox conditions. Both chemical
and biological oxidation/reduction reactions can occur. These
reactions often lead to dissolution and formation of iron minerals,
of which there are many. To understand the role of iron in biogeo-
chemistry, numerous studies have focused on assessing the effect
of environmental parameters on the redox conversion and mineral
dissolution/precipitation. Although advancements have been
made, there is still a large potential for future research. Compre-
hension of iron speciation and conversions lead to insights in the
effects of, for example, acid mine drainage and availability of
nutrients (e.g., Adams et al. 2007; Amstaetter et al. 2012; Behrends
and Van Cappellen 2007; Cooper et al. 2000; Wu et al. 2013). Fur-
thermore, the biological formation of minerals like magnetite is a
topic of high interest (e.g., Bharde et al. 2006; Bharde et al. 2005;
Byrne et al. 2015a; Ch^atellier et al. 2004; Dey et al. 2015).
Iron mineral phase identification is crucial for understanding
the role of iron in natural and engineered environments. Recently
we started research on iron mineral formation from ferrous iron
solutions, aiming to develop a new microbiological method for
iron removal from groundwater. In literature concerning microbi-
ological iron conversions, X-ray diffraction (XRD) is a commonly
used method for the phase identification of crystalline solids. In
many papers, also in the last decade, the most commonly applied
configuration for XRD uses a copper radiation source (e.g.,
Amstaetter et al. 2012; Behrends and Van Cappellen 2007; Bharde
et al. 2005, 2006; Ch^atellier et al., 2004; Cooper et al. 2000; Dey
et al. 2015; Wu et al. 2013). By using copper radiation when ana-
lyzing iron minerals however, it is often overlooked that the choice
of radiation should be compatible to the sample composition in
order to obtain a diffractogram of good quality. A mismatch
therein can lead to a high background signal that, especially in
combination with a low peak intensity, makes interpretation more
difficult and can even lead to incorrect phase identification. The
most striking example thereof is when dealing with magnetite
and/or maghemite. Diffractograms of these two minerals are only
distinguishable by a few low intensity peaks. Both can form in sim-
ilar environments and be converted into one another, in the case
of magnetite to maghemite simply by exposure to ambient air
(Cullity 1978).
The mismatch between copper radiation and iron is well-
known in the iron and steel industry, where cobalt radiation is
used (Klug and Alexander 1954), but is it not common knowl-
edge in other fields of research, while there is an increasing
interest in the biomineralization of iron oxides. This issue is
even more important in this field, where samples often do not
only contain crystalline, but also amorphous minerals and
microorganisms, which will increase the background signal fur-
ther. Despite indications in literature that it is unfavorable, cop-
per radiation is often used in XRD analysis of iron oxides and
interpretation of the results is not always sufficiently careful.
An example is found in Adams et al. 2007. The diffractograms
in Figure 6 and the interpretation thereof show that some peaks
that are used for identification are of equal height as the back-
ground noise. Other peaks are excluded from identification for
no apparent reason. A similar case is present in Byrne et al.

CONTACT Yvonne M. Mos yvonne.mos@wur.nl Sub-department of Environmental Technology, Wageningen University, Bornse Weilanden 9, 6708 WG,
Wageningen, The Netherlands.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ugmb.
© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
512 Y. M. MOS ET AL.
2015, Figures 1c and 4b, where peaks are hardly distinguishable
from the background and the identification of siderite is based
on the presence of only one peak. These diffractograms of mix-
tures of iron oxides were obtained with a copper radiation
source and are open to multiple interpretations. Drawing firm
conclusions from this data is therefore inadvisable. Many other
researchers draw conclusions based on diffractograms of iron
oxides obtained with copper radiation that are vulnerable for
incorrect interpretation (e.g., Behrends and Van Cappellen
2007; Bharde et al. 2005, 2006; Ch^atellier et al. 2004; Cooper
et al. 2000). Others strengthen their conclusions with TEM
analysis (Dey et al. 2015; Wu et al. 2013) or M€ ossbauer spec-
troscopy (Amstaetter et al. 2012). While this can confirm the
conclusions that are based on the XRD results, time, costs, and
effort can be saved by obtaining a good quality diffractogram in
the first place.
The incompatibility of copper radiation and iron oxides has
been indicated by, amongst others, Cullity, Fransen and Klug
and Alexander (Cullity 1978; Fransen 2004; Klug and Alexan-
der 1954). While these authors give a well-founded theoretical
explanation for this, examples and visuals to illustrate this are
not presented. In the current paper, we visualize the advantages
of cobalt radiation for the analysis of iron oxides as well as giv-
ing some other possible measures that can be taken to decrease
fluorescence.
In this paper, we demonstrate that the use of Cu radiation
for XRD analysis of iron oxides, as still applied in recent litera-
ture, can result in incorrect and/or incomplete phase identifica-
tion. We will illustrate this by analyzing four iron oxides which
are commonly investigated in biomineralization research (mag-
netite, goethite, maghemite, and hematite) with various equip-
ment configurations using both copper and cobalt radiation.
The results of the various scans are compared to show the
effects of the different configurations and radiation sources.
Theoretical background
The information in XRD analysis is provided by the interaction
between X-rays and the sample material. The different types of
interaction and their effects are discussed in this section, which
is primarily based on the works of Cullity (Cullity 1978).
When an X-ray hits an electron, as is the case in XRD imag-
ing, it will be scattered. The diffraction of the X-ray takes place
in all directions. Since in XRD a beam of X-rays (the incident
beam) hits a crystal lattice, many atoms are hit simultaneously.
The diffracted X-rays can either interact constructively or
destructively. This can be understood when one considers X-
rays to be waves; when in antiphase, the sum of the two waves
is zero. When the waves are in phase, they will reinforce each
other maximally, giving a high intensity peak in the
diffractogram.
X-rays have properties of both waves and particles (pho-
tons). Moreover, X-rays are electromagnetic, meaning that at
any point in a beam, there is a varying electric field. Scattering
can happen in two ways; elastic or inelastic. In elastic scattering,
the X-ray is diffracted by the electron it hits. Since the electron
is hit by a photon with an alternating electric field, it will start
to oscillate around its mean position. This causes the electron
to emit an electromagnetic wave of its own. This wave has the
same wavelength as the incident beam and therefore contrib-
utes to the diffractogram.
In inelastic scattering, the X-ray is also diffracted by the elec-
tron it hits. However, in this case, the X-ray knocks one of the
electrons out of the inner shell of an atom, emitting a character-
istic fluorescent radiation. While this is the basis of the tech-
nique of XRF (X-ray fluorescence), this is an unwanted
phenomenon in XRD. Since a relatively large part of the energy
of the incident beam is absorbed in this process, the intensity of
the beam reaching the detector (the diffracted beam), and
therefore the peak in the diffractogram, will be lower. Also, the
fluorescent radiation will be detected, causing a high back-
ground. These two effects combined can cause peaks to become
invisible, making it difficult to analyze the diffractogram.
Inelastic scattering and therefore fluorescence always takes
place during XRD. However, the amount of inelastic scattering
that occurs, depends on the combination of the type of X-rays
and the material that is analyzed. X-rays have a characteristic
radiation with peaks at different wavelengths, depending on the
source material. The wavelength of these peaks determines the
extent to which it will penetrate, absorb, and scatter. To ensure
sufficient scattering, the wavelength of the so called Ka radia-
tion peak used should be longer than the so called K absorption
edge of the sample material. The K adsorption edge of iron is
1.743A , which, in principle, would exclude copper (as well as
molybdenum) as suitable radiations for analysis (Table 1).
However, some physical measures can be taken to decrease the
background signal in the diffractogram. The first is setting a
sensitivity window for the detector. This window would typi-
cally be set for energies of the Ka § 50% keV (Table 1). This
solves the problem for iron analysis with molybdenum radia-
tion, but not for copper radiation (Cullity 1978; Fransen 2004).
Furthermore, one can use monochromators, both in the inci-
dent beam as well as in the diffracted beam (Fransen 2004). A
monochromator is a crystal like germanium or graphite. By
allowing the beam to be diffracted by the crystal, the radiation
of wavelengths other than Ka will be eliminated. In this way
the radiation hitting the sample as well as the radiation reach-
ing the detector can be ‘cleaned up’.
Applying a suitable type of radiation or using monochroma-
tors are both good options to avoid fluorescence (Cullity 1978;
Fransen 2004; Klug and Alexander 1954). However, as men-
tioned before, when a poorly suitable type of radiation is used,
a relatively large part of this will be absorbed by the sample.
This means that 1) besides causing a lower background signal,
it results in a lower peak intensity as well, and 2) it causes a
smaller penetration depth of the sample, implying that the vol-
ume of the sample that is analyzed in a scan is reduced, possibly
leading to a less representative result. Calculated penetration
Table 1. Wavelengths and characteristic energies of the Ka peak of different radia-
tions used in XRD.

Radiation Wavelength of Ka peak (A ) Characteristic Energy (keV)
Molybdenum 0.711 17.44
Copper 1.542 8.039
Cobalt 1.790 6.926
Iron 1.937 6.400
Manganese 2.103 5.895
Chromium 2.291 5.411

Figure 1. Calculated penetration depths of various types of radiation for
magnetite.
depths of different radiation types for magnetite are shown in
Figure 1.
Materials and methods
Four different iron oxides were purchased from chemical
suppliers. Indicated purity and particle size is according to
supplier information. The used magnetite (Fe3O4, Sigma
Aldrich, The Netherlands) is 97% pure, with a particle
size ranging from 50 to 100nm. Goethite (a-FeOOH, Santa
Cruz Biotechnology, USA) with a purity of 98% was used
(particle size not indicated). The maghemite (g-Fe2O3) was
obtained from US Research Nanomaterials, Inc. and is
99% pure, with nanoscale particle sizes ranging from 20
to 40nm. Lastly, the hematite (a-Fe2O3) (Cofermin Chemi-
cals GmbH & Co. KG, Germany) is 96% pure with a par-
ticle size of »700nm.
The iron oxide powders were analysed with a PANalyti-
cal Empyrean diffractometer system equipped with a sample
spinner and PreFIX (Pre-aligned Fast Interchangeable X-
ray) modules. The Bragg-Brentano diffraction geometry was
used in all cases. X-ray tubes with Cu and Co anode mate-
rials were utilized as radiation sources. Two different inci-
dent beam modules have been applied. First the traditional
incident beam slits (PDS, programmable divergence slits)
and secondly the novel Bragg-BrentanoHD module (BBHD),
which produces a monochromatized divergent incident
beam. The samples were spun with 1 rev/s. As detector the
PIXcel3D is used in linear (1D) scanning mode with adjust-
able energy sensitivity (PHD, pulse height distribution) set-
tings. The standard PHD settings used are set to a range of
25–80%, where the typical Ka energy of the radiation
source is defined at 50%. To suppress the fluorescence radi-
ation from Fe the lower level PHD setting was increased.
For the Cu radiation source the optimum peak-to-noise
ratio was found with a PHD range of 50–80%. For Co radi-
ation increasing the lower level PHD setting did not further
improve the peak-to-noise ratio. Finally the effectiveness to
remove fluorescence of a diffracted beam monochromator
was tested.
Scans were made over a 2u range of 10–110 with a total
exposure time of 15.5 min. For the regular powders a step size
of 0.0263 and a counting time of 56.9s/step were used. For the
GEOMICROBIOLOGY JOURNAL 513
nanocrystalline powder the step size and counting time were
adapted to 0.105 and 227s/step, respectively.
Results and discussion
Phase purity powder samples
The diffractograms gave insight into the phase purity of the
samples. Both the magnetite and maghemite samples had a
phase purity close to 100% based on the obtained diffracto-
grams. The goethite and hematite samples however, contained
various mineral phases. The goethite sample consisted of goe-
thite with both a cubic and an orthorhombic configuration.
Next to this, it contained about 40 to 50% hematite and 1 to 2%
quartz. The hematite sample contained about 10% maghemite
and a trace (1 – 2%) of quartz.
Qualifying the diffractogram
The peak to background ratio is commonly used for a compari-
son of diffractograms. This ratio is defined by the net peak
height of the highest peak divided by the background level
below that same peak:
Inet peak
P=B D (1)
Ibackground
A higher value for P/B indicates a better quality of the dif-
fractogram. This is a quick and easy evaluation of the quality.
However, this method does not take peak intensity loss into
account that is caused by physical measures taken to prevent
fluorescence (e.g., monochromators).
Considering the P/B ratio of the diffractograms of the mag-
netite sample for all tested configurations (Figure 2), the combi-
nation of Cu radiation with a diffracted beam monochromator
yields a good quality diffractogram. However, due to the loss of
efficiency, the net peak intensity is rather low. To counteract
this, the exposure time must be increased significantly (order of
magnitude 10 times) for small peaks to be visible. Hence, the
P/B ratio is not full proof as quality parameter in practice.
A different approach to qualify a diffractogram is looking at
the peak to noise ratio. Here, the net peak height of the highest
peak is divided by the noise of the background level. A com-
mon simplified formula for this ratio is obtained by replacing
Figure 2. Peak to background ratios of all tested configurations.
514 Y. M. MOS ET AL.

different configurations (Figure 3, magnetite sample). This is illus-

trated by the lower score of the setup with Cu radiation and the dif-
fracted beam monochromator. Therefore, we conclude that the
P/N ratio is an excellent measure to judge the quality of a
measurement.
Further improvement of the P/N ratio can be achieved by
increasing the exposure time. For example, to improve the
P/N ratio with a factor of 5, exposure time needs to be
25 times longer. Or alternatively, a measurement can be done
in a shorter time when a lower P/N ratio is considered as suf-
ficient. A commonly used rule of thumb is that the P/N ratio
has to be >3 to identify trace peaks and >10 for an accurate
phase quantification.
Figure 3. Peak to noise ratios of all tested configurations. Comparison of the diffractograms of maghemite measured
with PDS for the two radiation types shows that cobalt radia-
the latter by the square root of the number of counts of the tion gives both a lower background and a higher peak intensity
background level below that same peak: (Figures 4a and b). Comparison of the Cu and Co results of the
same sample with the highest P/N ratio however, shows a back-
Inet peak Inet peak
P=N D
 D pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi : (2) ground for the Cu radiation that is about 2.5 times lower
s Ibackground Ibackground (Figures 4c and d). However, due to the induced fluorescence
and the use of a BBHD, the intensity of the peaks is 5 times
With the P/N ratio, the loss of intensity is considered, making it lower. While the main peaks are visible in all diffractograms,
a more suitable parameter for comparison of diffractograms in the peaks with lower intensity (at larger d-spacing) are not

Figure 4. Diffractograms of the maghemite samples measured in the configurations a) Cu radiation with PDS; b) Co radiation with PDS; c) Cu radiation, BBHD and PHD 50
– 80; and d) Co radiation with BBHD.

detected in the diffractograms obtained with Cu radiation
(Figures 4a and c). The P/N ratio therefore, is better in the scan
with cobalt radiation.
The penetration depth of the radiation into the sample is
important for a representative result. This is especially true for
(inhomogeneous) multiphase samples or samples containing
impurities. The penetration depth or analysis volume is not
directly included in the P/N ratio. However, the X-ray intensity
yield is proportional to the analysed sample volume and there-
fore it is indirectly represented in the net peak intensity. Conse-
quently, we see no need to explore this further in this paper.
Nevertheless, when the occasion arises that one has to choose
between options with Cu or Co radiation with similar P/N
ratios, it is preferable to take the penetration depth of the radia-
tion type into account (Figure 1).
Implications for iron mineral identification
As the measurements with the Co – BBHD setup have the
best P/N ratio, these are used to provide an overview of the
different samples. Figure 5 shows the diffractograms of a)
magnetite, b) goethite, c) maghemite, and d) hematite. The
diffractograms of magnetite and maghemite in particular
Figure 5. Diffractograms of a) magnetite; b) goethite; c) maghemite; and d) hematite as measured with the Co – BBHD configuration.
GEOMICROBIOLOGY JOURNAL 515
show a great resemblance, as is highlighted in Figure 6. A
striking difference is the width of the peaks in case of the
maghemite. This is caused by the nanoscale particle size and
is independent of the type of iron oxide and is therefore not
regarded as a relevant parameter in this comparison. It is
clearly visible that both materials share many peaks at the
same d-spacing. The distinguishing peaks, specifically at high
d-spacing (left side of X-axis), are small. In the scan with Co
– BBHD, there are clearly distinguishable peaks present for
the maghemite sample in this range, while in the Cu – BBHD
– PHD50-80 scan, these are obscured by the background (see
frame). Moreover, the latter shows a shoulder for the highest
intensity peak. This could be mistaken for one of the signa-
ture peaks of magnetite that occurs around the same d-spac-
ing. When considering a single type of sample of either
mineral with this configuration therefore, the identification
becomes inconclusive. When a sample is measured with a
shorter exposure time, the smaller peaks will become even
less distinguishable. With only the larger intensity peaks visi-
ble, a maghemite sample can easily be identified as magnetite.
Moreover, these analysis have been performed on high grade
chemical iron oxides, many samples in research are of a lower
grade and/or are mixtures of several phases, complicating
516 Y. M. MOS ET AL.
Figure 6. Comparison of diffractograms of the magnetite and maghemite samples as measured with a) Co – BBHD; and b) Cu – BHHD – PHD50–80 configurations. Frames
highlight the distinguishing peaks.
analysis further. A good quality diffractogram is therefore
vital for correct phase identification.
Conclusion
Analyzing an iron containing sample with copper radiation yields
a high background level due to fluorescence. This can lead to
incorrect phase identification, especially when dealing with mag-
netite, maghemite or mixtures. Multiple measures can be applied
to suppress fluorescence (e.g., monochromators). However, these
also have disadvantages, like loss of peak intensity and low pene-
tration depth, leading to diffractograms that yield inconclusive
identification. This is avoided by applying cobalt radiation. The
lower fluorescence leads to a superior peak to noise ratio and
therefore an unambiguous phase identification. We recommend
that for the XRD analysis of iron oxides, cobalt radiation is used
to avoid misinterpretations. In addition, research reported in the
past that made use of copper radiation for this type of samples
without special measures, should be interpreted cautiously.
Funding
This research is supported by the Dutch Technology Foundation STW,
which is part of the Netherlands Organisation for Scientific Research
(NWO), and which is partly funded by the Ministry of Economic
Affairs.
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